The invention relates to bacterial and bacteriophage genes.
The Staphylococci make up a medically important genera of microbes known to cause several types of diseases in humans. S. aureus is a Gram positive organism which can be found on the skin of healthy human hosts. It is responsible for a large number of bacteremias, where its portal of entry can be the skin, lungs, urinary tract or infected intravascular devices (Steinberg et al., (1996)) Clin. Infect. Dis. 23: 255-259; Rxc3x8der et al., (1999) Arch. Intern. Med. 159: 462-469). It can cause fatal endocarditis or damage to the heart and, due to its exotoxin, can cause death via xe2x80x9cToxic Shockxe2x80x9d (Frimodt-Mxc3x8ller et al., (1997) Clin. Microbiol. Infect. 3: 297-305; Sanabria et al., (1990) Arch. Intern. Med. 150: 1305-1309).
Only S. aureus and Staphylococcus epidermidis, of the nineteen species of Staphylococcus described in Bergey""s Manual (1992), have significant interactions with humans. They are among the normal flora of humans, and are found on nasal passages, skin and mucous membranes. S. aureus, when pathogenic in humans, can cause a number of suppurative (pus-forming) infections, as well as food poisoning, endocarditis, and toxic shock syndrome.
S. aureus causes superficial skin lesions, such as boils, styes and furunculosis; more serious infections such as pneumonia, mastitis, phlebitis, meningitis, and urinary tract infections, in addition to osteomyelitis and endocarditis. S. aureus is also a major cause of hospital acquired (nosocomial) infection of surgical wounds and infections associated with inserted and implanted medical devices. Lastly, S. aureus causes food poisoning through the release of enterotoxins into food, and toxic shock syndrome through the release of superantigens into the blood stream. S aureus also secretes two types of toxin with superantigen activity: 1) enterotoxins, of which there are six antigenic types (named SE-A, B, C, D, E and G) and 2) toxic shock syndrome toxin (TSST-1).
S. aureus has been successfully treated with the penicillin derivative Methicillin in the past, but is now becoming increasingly resistant (MRSAxe2x80x94Methicillin Resistant S. aureus) to this antibiotic (Harbath et al., (1998) Arch. Intern. Med. 158: 182-189.). For example, S. aureus endocarditis mortality can range from 26-45%, and combined xcex2-lactam/aminoglycoside therapy is proving increasingly ineffective in disease eradication (Rxc3x8der et al., (1999) Arch. Intern. Med. 159: 462-469). However, MRSA infections continued to be sensitive to treatment with vancomycin which is the drug of last resort. Infections caused by MRSA have been increasing in children and adults; isolates have been found in 97% of all large, university-based teaching hospitals in the United States. Since 1996, three cases of vancomycin resistant S. aureus have been reported. This new strain represents a particularly dangerous development of an aggressive bacterial pathogen which does not respond to any known antibiotic. The emergence of resistance to vancomycin has the potential to result in untreatable (and thus fatal) S. aureus infections.
It is no longer uncommon to isolate S. aureus strains which are resistant to most of the standard antibiotics, and thus there is an unmet medical need and demand for new anti-microbial agents, vaccines, drug screening methods, and diagnostic tests for this organism.
The present invention relates to dnaI and dnaI related proteins, in particular S. aureus DnaI polypeptides and dnaI polynucleotides, recombinant materials and methods for their production. The invention also relates to a pair of interacting proteins, a growth-inhibitory (or killer) bacteriophage P77 ORF 104 gene product that interacts with the S. aureus DnaI polypeptide, the interacting regions of the S. aureus DnaI related protein and the protein encoded by the S. aureus bacteriophage 77 ORF 104, forming the basis for screening assays. It also relates to polynucleotides and polypeptides of a multiprotein complex believed to be involved in initiation of DNA replication containing DnaI as a subunit, and also may include DnaC and related proteins, as well as variants of them. In another aspect, the invention relates to methods for using such polypeptides and polynucleotides, including treatment of microbial diseases, amongst others. In a further aspect, the invention relates to methods for identifying agonists and antagonists using the materials provided by the invention, and for treating microbial infections and conditions associated with such infections with the identified agonist or antagonists compounds. In a still further aspect, the invention relates to diagnostic assays for detecting diseases associated with microbial infections and conditions associated with such infections, such as assays for detecting dnai expression or activity.
The invention encompasses a method of identifying a compound that is active on a S. aureus DnaI polypeptide, comprising contacting a candidate compound with the DnaI polypeptide, and detecting binding of the candidate compound to the DnaI polypeptide, wherein detection of binding is indicative that the compound that is active on the DnaI polypeptide.
In one embodiment, the step of detecting comprises the step of measuring the binding of a candidate compound, wherein the compound is directly or indirectly detectably labeled, to the DnaI polypeptide.
In another embodiment, the step of detecting comprises measurement by surface plasmon resonance.
In another embodiment, the step of detecting comprises measurement by FRET.
In another embodiment, the step of detecting comprises measurement of fluorescence polarization changes.
In another embodiment, the step of detecting comprises a scintillation proximity assay.
In another embodiment, the step of detecting comprises a biosensor assay.
The invention further encompasses a method of identifying a compound that is active on a DnaI polypeptide, comprising the steps of contacting a candidate compound with cells expressing a DnaI polypeptide and detecting DnaI polypeptide activity in the cells, wherein a decrease in activity relative to DnaI activity in cells not contacted with a candidate compound is indicative of inhibition of DnaI activity.
In one embodiment, the step of detecting comprises measuring the incorporation of 3H-thymidine into DNA.
In another embodiment, the step of detecting comprises measuring plasmid replication.
The invention further encompasses an agonist or an antagonist of the activity of a DnaI polypeptide or a gene encoding the polypeptide.
The invention further encompasses a method of identifying a compound that is active on a polypeptide complex comprising a S. aureus DnaI polypeptide and a polypeptide of S. aureus DnaC, the method comprising: providing the polypeptide complex and a candidate compound; and detecting an increase or decrease in the amount of the complex in the presence of the candidate compound, wherein an increase or decrease in the amount in the presence of the candidate compound relative to its absence is indicative of a compound that is active on the complex.
In one embodiment, the providing step comprises permitting said polypeptide complex to form in the presence of the candidate compound.
The invention further encompasses a method of making an antibacterial compound, comprising the steps of: i) determining whether a candidate compound is active on a DnaI polypeptide or a gene encoding the polypeptide; and ii) synthesizing or purifying the candidate compound in an amount sufficient to provide a therapeutic effect when administered to an organism infected by a bacterium naturally producing the polypeptide.
In one embodiment, the candidate compound is selected from the group consisting of a small molecule, a peptidomimetic compound, and a fragment or derivative of a bacteriophage inhibitor protein.
The invention further encompasses a method for inhibiting a bacterium, comprising contacting the bacterium with a compound active on a S. aureus DnaI polypeptide or a gene encoding the polypeptide.
In one embodiment, the step of contacting is performed in vitro.
In another embodiment, the step of contacting is performed in vivo in an animal.
The invention further encompasses a method for treating a bacterial infection in an animal suffering from an infection, comprising administering to the animal a therapeutically effective amount of a compound active on a S. aureus DnaI polypeptide or a gene encoding the polypeptide.
In one embodiment, the compound is selected from the group consisting of a small molecule, a peptidomimetic compound, and a bacteriophage inhibitor protein.
The invention further encompasses a method of prophylactic treatment to prevent bacterial infection comprising contacting an indwelling device with a compound active on a S. aureus DnaI polypeptide before its implantation into a mammal, such contacting being sufficient to prevent S. aureus infection at the site of implantation.
The invention further encompasses a method of prophylactic treatment to prevent infection of an animal by a bacterium comprising administering to the animal a compound that is active on a S. aureus DnaI polypeptide or a gene encoding the polypeptide in an amount sufficient to reduce adhesion of the bacterium to a tissue surface of a tissue of the mammal.
The invention further encompasses a method of diagnosing in an individual an infection with Staphylococcus aureus, comprising: determining the presence in the individual of a S. aureus DnaI polypeptide.
In one embodiment, the determining step comprises contacting a biological sample of the individual with an antibody specific for an epitope present on a S. aureus DnaI polypeptide.
The invention further encompasses a method of diagnosing in an individual an infection with Staphylococcus aureus, comprising: determining the presence in the individual of a nucleic acid sequence encoding a S. aureus DnaI polypeptide.
In one embodiment, the determining step comprises contacting a nucleic acid sample of said individual with an isolated, purified or enriched nucleic acid probe of at least 15 nucleotides in length that hybridizes under stringent hybridization conditions with the sequence of SEQ ID NO:1, or the complement of such probe.
The invention further encompasses an isolated, purified or enriched polynucleotide comprising a nucleotide sequence that has at least 60% identity to the sequence of SEQ ID NO:1, or the complement of said nucleotide sequence.
The invention further encompasses an isolated, purified or enriched polynucleotide comprising a nucleotide sequence encoding the polypeptide of SEQ ID NO:2, or the complement of such nucleotide sequence.
The invention further encompasses an isolated, purified or enriched polynucleotide comprising the polynucleotide of SEQ ID NO:1, or the complement of said polynucleotide of SEQ ID NO:1.
The invention further encompasses an isolated, purified or enriched polynucleotide of at least 15 nucleotides in length having at least 80% identity to a sequence selected from the group consisting of nucleotides 1-113, 101-496, 484-529, 518-538, 526-548, 551-606, 596-614, 602-693, 694-736, 736-791, 780-794, and 791-942, inclusive, of SEQ ID NO:1, or the complement of such a nucleotide sequence.
The invention further encompasses an isolated, purified or enriched polynucleotide of at least 16 nucleotides in length having at least 81% identity to a sequence selected from the group consisting of nucleotides 1-530, 517-549, 540-555, 550-607, 595-694, 693-737, 729-744, 735-795, and 790-942, inclusive, of SEQ ID NO:1, or the complement of such a nucleotide sequence.
The invention further encompasses an isolated, purified or enriched polynucleotide of at least 16 nucleotides in length having at least 75% identity to a sequence selected from the group consisting of nucleotides 1-68, 55-103, 90-111, 102-122, 112-496, 484-502, 489-529, 519-538, 528-543, 530-547, 551-603, 597-613, 602-691, 697-736, 736-790, 791-828, 816-832, and 820-942, inclusive, of SEQ ID NO:1, or the complement of such a nucleotide sequence.
The invention further encompasses an isolated, purified or enriched polynucleotide of at least 17 nucleotides in length having at least 76% identity to a sequence selected from the group consisting of nucleotides 1-112, 99-115, 101-123, 111-530, 518-540, 527-548, 550-604, 596-614, 600-692, 696-737, 735-791, 777-794, 790-829, 815-833, and 819-942, inclusive, SEQ ID NO:1, or the complement of such a nucleotide sequence.
The invention further encompasses an isolated, purified or enriched polynucleotide of at least 17 nucleotides in length having at least 70% identity to a sequence selected from the group consisting of nucleotides 1-68, 56-97, 83-100, 86-103, 91-107, 102-121, 113-200, 186-496, 485-501, 489-521, 507-526, 512-529, 551-598, 584-601, 587-603, 602-691, 697-718, 704-736, 740-757, 743-767, 753-790, 791-828, 816-832, 820-877, and 863-942, inclusive, SEQ ID NO:1, or the complement of such a nucleotide sequence.
The invention further encompasses an isolated, purified or enriched nucleic acid probe of at least 15 nucleotides in length that hybridizes under stringent hybridization conditions with the sequence of SEQ ID NO:1, or the complement of the probe.
The invention further encompasses an isolated, purified or enriched polynucleotide consisting of the sequence of SEQ ID NO:1.
The invention further encompasses an isolated, purified or enriched polypeptide having at least 50% identity to the amino acid sequence of SEQ ID NO:2.
The invention further encompasses an isolated, purified or enriched polypeptide of at least 5 amino acids in length having at least 80% identity to a sequence selected from the group consisting of amino acids 1-23, 21-167, 171-176, 174-184, 185-202, 200-204, 202-231, 235-243, 247-262, 266-275, 273-290, 289-308, 306-310, and 308-313, inclusive, of SEQ ID NO:2.
The invention further encompasses an isolated, purified or enriched polypeptide of at least 6 amino acids in length having at least 66% identity to a sequence selected from the group consisting of amino acids 1-23, 22-41, 38-62, 60-108, 105-167, 175-180, 177-183, 185-201, 204-231, 236-243, 248-262, 266-275, 273-289, 291-308, and 308-313, inclusive, of SEQ ID NO:2.
The invention further encompasses an isolated, purified or enriched polypeptide of at least 6 amino acids in length having at least 83% identity to a sequence selected from the group consisting of amino acids 1-168, 170-185, 182-187, 184-232, 234-244, 246-263, and 265-313, inclusive, of SEQ ID NO:2.
The invention further encompasses an isolated, purified or enriched polypeptide of at least 7 amino acids in length having at least 57% identity to a sequence selected from the group consisting of amino acids 1-23, 22-28, 24-37, 33-40, 38-58, 54-62, 60-66, 62-68, 64-104, 100-106, 102-108, 106-131, 127-167, 186-200, 204-222, 218-231, 248-260, 268-275, 273-288, and 291-307, inclusive, of SEQ ID NO:2.
The invention further encompasses an isolated, purified or enriched polypeptide of at least 7 amino acids in length having at least 71% identity to a sequence selected from the group consisting of amino acids 1-25, 21-63, 59-168, 172-178, 174-184, 184-202, 198-204, 200-206, 203-232, 235-244, 247-263, 265-290, 286-293, 290-310, and 306-313, inclusive, SEQ ID NO:2.
The invention further encompasses an isolated, purified or enriched polypeptide of at least 8 amino acids in length having at least 50% identity to a sequence selected from the group consisting of amino acids 1-23, 24-37, 33-40, 38-58, 55-62, 64-91, 86-103, 106-131, 127-167, 186-200, 204-219, 214-222, 218-231, 250-260, 273-288 and 291-307, inclusive SEQ ID NO:2.
The invention further encompasses an isolated, purified or enriched polypeptide of at least 8 amino acids in length having at least 62% identity to a sequence selected from the group consisting of amino acids 1-25, 21-41, 36-63, 59-110, 105-168, 175-182, 185-201, 203-232, 247-261, 267-289, 286-293, 290-308, 303-310, and 306-313, inclusive, of SEQ ID NO: 2.
The invention further encompasses an isolated, purified or enriched polypeptide of at least 8 amino acids in length having at least 75% identity to a sequence selected from the group consisting of amino acids 1-169, 172-185, 180-187, 182-207, 202-233, 234-245, 246-264, 264-294, and 289-313, inclusive, of SEQ ID NO:2.
The invention further encompasses an isolated, purified or enriched polypeptide having at least 70% similarity to the amino acid sequence of SEQ ID NO:2.
The invention further encompasses an isolated, purified or enriched polypeptide of at least 20 amino acids in length having at least 60% similarity to the amino acid sequence of SEQ ID NO:2.
The invention further encompasses an isolated, purified or enriched polypeptide of at least 9 amino acids in length having at least 44% sequence similarity to a sequence selected from the group consisting of amino acids 2-11, 8-21, 43-54, 65-80, 94-102, and 141-166, inclusive, of SEQ ID NO:2.
The invention further encompasses an isolated, purified or enriched polypeptide of at least 10 amino acids in length having at least 50% sequence similarity to a sequence selected from the group consisting of amino acids 1-13, 6-22, 42-55, 64-82, 91-100, 93-103, 129-138, 133-143, 140-167, and 297-306, inclusive, of SEQ ID NO:2.
The invention further encompasses an isolated, purified or enriched polypeptide of at least 20 amino acids in length having at least 50% sequence similarity to a sequence selected from the group consisting of amino acids 1-23, 57-84, 68-87, and 129-170, inclusive, of SEQ ID NO:2.
The invention further encompasses an isolated, purified or enriched polypeptide of at least 20 amino acids in length having at least 60% sequence similarity to a sequence selected from the group consisting of amino acids 1-27, 32-55, 38-66, 54-91, 87-106, 89-113, 115-134, 117-136, 123-175, 208-228, and 268-288, inclusive, of SEQ ID NO:2.
The invention further encompasses an isolated, purified or enriched polypeptide of at least 20 amino acids in length having at least 70% sequence similarity to a sequence selected from the group consisting of amino acids 1-30, 29-127, 110-129, 112-178, 192-212, 203-230, 263-290, 285-290, 287-306, and 291-313, inclusive, of SEQ ID NO:2.
The invention further encompasses an isolated, purified or enriched polypeptide of at least 25 amino acids in length having at least 48% sequence similarity to a sequence selected from the group consisting of amino acids 127-173, inclusive, of SEQ ID NO:2.
The invention further encompasses an isolated, purified or enriched polypeptide of at least 25 amino acids in length having at least 60% sequence similarity to a sequence selected from the group consisting of amino acids 1-28, 33-58, 36-60, 38-98, 76-100, 86-111, 91-115, 93-119, 114-139, and 117-178, inclusive, of SEQ ID NO:2.
The invention further encompasses an isolated, purified or enriched polypeptide of at least 25 amino acids in length having at least 68% sequence similarity to a sequence selected from the group consisting of amino acids 1-32, 10-34, 27-126, 105-180, 158-182, 187-214, 192-217, 202-233, 263-293, 277-301, and 282-306, inclusive, of SEQ ID NO:2.
The invention further encompasses an isolated polypeptide comprising the amino acid sequence of SEQ ID NO:2.
The invention further encompasses an isolated polypeptide consisting of the amino acid sequence of SEQ ID NO:2.
The invention further encompasses an isolated, purified or enriched antibody specific for a polypeptide of the invention.
The invention further encompasses a composition comprising two polypeptides, a bacteriophage P77 ORF 104-encoded polypeptide and a S. aureus DnaI polypeptide.
The invention further encompasses a composition comprising at least three polypeptides, a bacteriophage P77 ORF 104-encoded polypeptide, a S. aureus DnaI polypeptide and a S. aureus DnaC polypeptide.
The invention further encompasses a composition comprising at least two polypeptides, a S. aureus DnaI polypeptide and a S. aureus DnaC polypeptide.
The invention further encompasses a composition comprising two nucleic acid sequences, bacteriophage P77 ORF 104 and S. aureus dnaI nucleic acid sequence.
The invention further encompasses a composition comprising at least two nucleic acid sequences, a S. aureus dnaI nucleic acid sequence and a S. aureus dnaC nucleic acid sequence.
The invention further encompasses a composition comprising at least three nucleic acid sequences, bacteriophage P77 ORF 104 nucleic acid sequence , a S. aureus dnaI nucleic acid sequence, and a S. aureus dnaC nucleic acid sequence.